Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity

We have suggested previously that the amino-terminal 8 kilodaltons of pp60^src may serve as a structural hydrophobic domain through which pp60^src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60^src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, in contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60^src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60^src, and suggest that the amino terminus of pp60^src may have a critical role in the promotion of in vivo tumorigenicity.     

Use of a synthetic DNA oligonucleotide to probe the precision of RNA splicing in a yeast mitochondrial petite mutant.

In some strains of Saccharomyces cerevisiae the mitochondrial gene coding for 21S rRNA is interrupted by an intron of 1143 bp. This intron contains a reading frame for 235 amino acids: Unassigned Reading Frame (URF). In order to check whether expression of this URF is required for proper splicing of precursors to 21S rRNA, the precision of RNA splicing was analysed in a petite mutant, where no mitochondrial protein synthesis is possible anymore. We have devised a new assay to monitor the precision of the splicing event. The method is of general application, provided that the sequence of the splice boundaries is known. In the case of the 21S rRNA it involves the synthesis of the DNA oligonucleotide d(CGATCCCTATTGTC( complementary to the 5' d(CGATCCCTAT) and 3' d(TGTC) borders flanking the intron in the 21S rRNA gene. The oligonucleotide is labelled with 32p at the 5'-end, hybridised to RNA and subsequently subjected to digestion with S1 nuclease. Resistance to digestion will only be observed if the correct splice-junction is made. The petite mutant we have studied contains a 21S rRNA with the same migration behaviour as wildtype 21S rRNA. In RNA blotting experiments, using an intron specific hybridisation probe, the same intermediates in splicing are found both in wild type and petite mutant. Finally the synthetic oligonucleotide hybridises to petite 21S rRNA and its thermal dissociation behaviour is indistinguishable from a hybrid formed with wildtype 21S rRNA. We conclude that expression of the URF, present in the intron of the 21S rRNA gene, is not required for processing and correct splicing of 21S ribosomal precursor RNA.     

Aspects of amino acid requirements for in vitro evagination of imaginal leg discs inDrosophila melanogaster

Summary A method of staging late third instar larvae on the basis of salivary gland morphology is described. Using this technique, we investigated stage related amino acid requirements forDrosophila leg disc evagination in vitro. It was found that the requirement for glutamine lasted longer than that of proline. The staging technique should help in the detailed exploration of the late 3rd instar time period in order to bridge the gap between biochemistry and morphogenesis in the initiation of disc evagination.     

Effect of the TP5 analogue of thymopoietin on the rejection of male skin by aged and thymectomized female mice

Although young adult C3H/HeJ (OH) females do not reject C3H male skin grafts, OH females older than 1 year commonly do so, as also do many thymectomized, young adult C3H females. Therapy With TP5, a synthetic pentapeptide analogue of thymopoietin which has biological properties of the parent molecule, substantially reduced the capacity of aged OH females and of thymectomized, young OH females to reject OH male skin.     

Association of long surface appendages with adherence-related functions of the gram-positive species Actinomyces naeslundii

Electron microscopy of new isolates of gram-positive Actinomyces naeslundii demonstrated long, fragile appendages. Removal of the appendages impaired attachment to epithelial cells and reaggregation, thus implicating them in attachment-related functions.     

Cinnamic acid 4-hydroxylase from gherkin tissues

Homogenates from 4-day-old gherkin cotyledons and hypocotyls fractionated by sucrose density gradient centrifugation contain cinnamic acid 4-hydroxylase activity, the activity being highest in the endoplasmic reticulum fractions. These fractions also contain very low concentrations of cytochrome P₄₅₀. Hydroxylase activity is dependent on NADPH and on molecular oxygen, is optimal at pH 7.5 and is inhibited by carbon monoxide. The enzyme is very sensitive to inhibition by 2-mercaptoethanol, but it is not inhibited by the product, p-coumaric acid. Further, its responses to various potential inhibitors are fairly typical of mixed function oxidases from other sources.     

Distribution and seasonality ofLegionella pneumophila in colling towers

The numbers of presumptiveLegionella pneumophila cells in waters and sediments of nine different cooling towers located on the same site in the northeastern United States were determined at approximately monthly intervals for 18 months. All systems received makeup water from the same source and received the same chemical treatments. PresumptiveL. pneumophila were found in both water and sediment samples from all systems on all sampling dates. An important result of this study was the finding that tower sediments represent large reservoirs ofL. pneumophila. The only correlation between levels of presumptiveL. pneumophila and any of the physical, chemical, or operating characteristics evaluated was with winter shutdown and drainage followed by a nonoperational period. These systems showed a definite seasonal response with the highest levels of presumptiveL. pneumophila found in the summer and fall. Systems operated year round showed relatively constant numbers ofL. pneumophila in both water and sediments.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species.